Angelman Syndrome is rare but severe neurodevelopmental disorder characterized by intellectual disability, motor impairment, and happy demeanors. Genetic cause of the disease is the deletion/abnormal expression of maternal chromosome 15q11-q13, which includes the UBE3A gene that codes for the E6-associated protein (E6-AP). A mouse model (AS mice) of the human disease has been generated by deletion of maternal Ube3a; these mice exhibit impaired long-term potentiation (LTP) of synaptic transmission in hippocampus, learning of various hippocampus-dependent tasks, and motor functions. Preliminary results indicated that abnormal calpain-2 activity might contribute to synaptic and cognitive impairment in AS mice, as treatment with a calpain-2 inhibitor restored normal LTP in slices prepared from AS mice. The rationale for the preliminary study was based on our recent discovery that calpain-2 activation during LTP consolidation functions as a molecular brake that limits the magnitude of long-term potentiation in hippocampus. A major component of the molecular brake is calpain-2-mediated degradation of the tumor suppressor PTEN, and AS mice have enhanced calpain-2 expression and decreased PTEN levels in hippocampus. These results suggest that calpain-2 might be a good target to restore normal learning in the Angelman Syndrome. While we have identified the dipeptide ketoamide, Z-Leu-Abu-CONH-CH2-C6H3(3,5-(OMe)2) as a relatively selective calpain-2 inhibitor at low concentrations, at higher concentrations it also inhibits calpain-1, which is necessary for LTP induction. As PTEN is selectively cleaved by calpain-2 and not calpain-1, we posit that identifying PTEN properties underlying its selectivity for calpain-2 will provide criticl information to develop calpain-2 selective inhibitors, which will not inhibit calpain-1. The proposed studies are directed at using detailed molecular dynamics simulation to identify critical features in PTEN that account for calpain-2 selectivity. We will then use a multi-level virtual screening method to design new and selective calpain- 2 inhibitors. These inhibitors will be tested first on in vitro assays for calpain-1 and -2 and for interactions with other cysteine proteases and then for their effects on LTP and in learning in the AS mice. Successful design and testing of a selective calpain-2 inhibitor will then lead to a potential U01 submission for using such inhibitors not only for AS treatment but also for other indications with learning and memory impairment.